There has been a surge in interest in the ocean as a source of new therapeutics {Blunt et al., 2012, Nat Prod Rep, 29, 144-222}. This has in part been stimulated by high profile successes and by belief that the less well-explored marine environment contains many more unexploited resources {Driggers et al., 2008, Nat Rev Drug Discov, 7, 608-24; Mayer et al., 2013, Mar Drugs, 11, 2510-73}. Ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by marine organisms have been shown to possess anti-tumour, anti-fungal, antibacterial and antiviral properties {Sivonen et al., 2010, Appl Microbiol Biotechnol, 86, 1213-25}. Cyanobactins, peptide derived natural products from cyanobacteria, are RiPPs in which one or more core peptides (it is the core peptide which becomes a natural product) are embedded into a larger precursor peptide. The most well-known example of this class are the patellamides, whose biosynthetic pathway was one of the first cyanobactin pathways to be described and cloned {Schmidt et al., 2005, Proc Natl Acad Sci USA, 102, 7315-20; Donia et al., 2006, Nat Chem Biol, 2, 729-35; Long et al., 2005, Chembiochem, 6, 1760-5}. The precursor peptide has an N-terminal leader, typically around 40 residues, which is disposed of during maturation {Arnison et al., 2013, Nat Prod Rep, 30, 108-60}. Characterized modifications of the core peptide are extensive and include heterocyclization of Ser/Thr and Cys residues to oxazolines and thiazolines, oxidation of these heterocycles to oxazoles and thiazoles, epimerization of amino acids to give D-stereocenters, Ser/Thr/Tyr prenylation and macrocycle formation {Milne et al., 2006, Org Biomol Chem, 4, 631-8; Schmidt et al., 2005, Proc Natl Acad Sci USA, 102, 7315-20; Schmidt and Donia, 2009, Methods Enzymol, 458, 575-96}. The permissiveness of the modifying enzymes to sequence changes in the core peptide has been demonstrated by the creation of large libraries of novel macrocycles made in vivo by genetic engineering {Donia et al., 2006, Nat Chem Biol, 2, 729-35; Donia and Schmidt, 2011, Chem Biol, 18, 508-19}. Of the enzymes, which have been structurally and biochemically characterized, three, the protease (which cleaves off the leader), the heterocyclase and macrocyclase, recognize regions outside the core peptide to accomplish their transformations {Houssen et al., 2012, Chembiochem, 13, 2683-9; Koehnke et al., 2012, Nat Struct Mol Biol, 19, 767-72; Koehnke et al., 2013, Chembiochem, 14, 564-7; Koehnke et al., 2013, Angew Chem Int Ed Engl, 52, 13991-6; Agarwal et al., 2012, Chem Biol, 19, 1411-22}. The recognition beyond the functional group that governs the prenylase {Bent et al., 2013, Acta Crystallogr Sect F Struct Biol Cryst Commun, 69, 618-23; Majmudar and Gibbs, 2011, Chembiochem, 12, 2723-6}, oxidase {Melby et al., 2014, Biochemistry, 53, 413-22} and hypothetical epimerase remain unknown.
The first chemical transformation in the biosynthesis of the patellamides is the heterocyclization of core peptide Cys (and sometimes Ser/Thr) residues to thiazolines (and oxazolines) {McIntosh and Schmidt, 2010, Chembiochem, 11, 1413-21}. The site-selective introduction of heterocycles into peptide backbones alters both conformation and reactivity of peptides; this tailoring of peptides is highly desirable in modifying their biological properties {Nielsen et al., 2014, Angew Chem Int Ed Engl}. This step is carried out by a conserved class of ATP and Mg2+-dependent YcaO-domain containing heterocyclases, exemplified by the enzymes PatD and TruD from the patellamide and trunkamide pathways, respectively {McIntosh and Schmidt, 2010, Chembiochem, 11, 1413-21; McIntosh et al., 2010, J Am Chem Soc, 132, 4089-91}.
The recognition elements that control the substrate processing was not known although the N-terminal leader of substrate peptides is required for processing by TruD/PatD {McIntosh and Schmidt, 2010, Chembiochem, 11, 1413-21; McIntosh et al., 2010, J Am Chem Soc, 132, 4089-91} but no molecular insight has been forthcoming. The apo structure of the cyanobactin heterocyclase TruD was reported and showed this enzyme to be a three-domain protein {Koehnke et al., 2013, Angew Chem Int Ed Engl, 52, 13991-6}. The first two domains share structural but limited sequence homology with MccB (an adenylating enzyme from the microcin pathway) {Regni et al., 2009, EMBO J, 28, 1953-64} and the third domain (the ‘YcaO’ domain) had, at that time, no homology to known structures {Koehnke et al., 2013, Angew Chem Int Ed Engl, 52, 13991-6}.
Analysis of both the BalhD and TruD heterocyclases has shown they operate with a preferred order, starting at the C-terminus {Melby et al., 2012, J Am Chem Soc, 134, 5309-16; Koehnke et al., 2013, Angew Chem Int Ed Engl, 52, 13991-6}. By a series of deletions and site directed mutants of and within the PatE leader peptide the substrate recognition motif was narrowed (denoted ‘minimal leader’) {Koehnke et al., 2013, Angew Chem Int Ed Engl, 52, 13991-6}. It was also shown that TruD was able to process the C-terminal cysteine of test peptides which lacked the leader, albeit more slowly, but TruD was not, within the timescale of the experiment, able to process a second ‘internal’ cysteine {Koehnke et al., 2013, Angew Chem Int Ed Engl, 52, 13991-6}. A recent study has reported that trans activation of the PatD enzyme by exogenous leader peptide restored processing activity for internal cysteines {Goto et al., 2014, Chem Biol, 21, 766-74}.
WO2014136971 reports the production of compounds containing heterocyclic rings using heterocyclases linked to leader sequences. However, the reaction is inefficient and generates multiple products containing different numbers of heterocyclic residues.